In human pathogen Pseudomonas aeruginosa, quorum sensing (QS) is crucial in regulating the expression of a large number of genes, especially those encoding virulence factors. Consequently, there is a need to understand how QS is regulated. QS systems in P. aeruginosa consist of the Las and Rhl systems and these two systems are also linked to Pseudomonas aeruginosa quinolone signal (PQS) signalling systems. It has already been reported that QS is regulated at the transcriptional and post-transcriptional level by many factors, but how QS threshold is modulated remains obscure. In this study, a genetic screen of transposon mutants with changed QS phenotypes was carried out and this led to the discovery of anti-activators QslA and QslH.
In qslA null mutant, there was enhanced PQS signalling and greater production of virulence factors compared to wild type. Conversely, overexpression of QslA abolished QS, PQS signalling and virulence factor production. QslA was determined to inhibit LasR post-transcriptionally and it was found using co-immunoprecipitation analysis that QslA inhibited QS by protein-protein interaction with LasR. Electrophoretic mobility shift analysis (EMSA) analysis showed that QslA disrupted LasR activation of gene expression by impeding LasR binding to DNA.
In addition to its control of QS response, QslA also influenced the QS activation threshold. In qslA mutant, 9 times less QS signals was sufficient to activate QS-dependent virulence factor production. This finding indicates that QslA is responsible for raising the QS ¿threshold hurdle¿ so that QS is activated at a high QS threshold concentration or a high bacterial cell density.
In the same transposon mutant screen, another anti-activator named QslH, was also identified. When qslH was overexpressed, QS and PQS signalling systems as well as virulence factor production were inhibited. Using co-immunoprecipitation analysis, QslH was found to interact with LasR and PqsR. QslH interaction with LasR was verified by studying LasR activity in Escherichia coli and by EMSA. Results from bacterial two-hybrid analysis also confirmed that QslH interacts with PqsR.
Null mutant of qslH did not differ from the wild type in its QS and PQS signalling phenotype. Thus, QslH inhibited QS and PQS signalling only when overexpressed. Hence, it was hypothesized that QslH was not expressed at adequately high levels to affect QS and PQS signalling in the wild type under the experimental conditions used in this study. Transposon mutagenesis was carried out and it was found that qslH expression was increased in the absence of mvaT. In mvaT mutant, enhanced level of QslH inhibited production of virulence factors. Taken together, the results demonstrate that QslH plays a role in QS regulation at the downstream of MvaT-dependent regulatory networks.
Identification of QslA and QslH demonstrates that QS activation in P. aeruginosa is modulated by protein-protein interaction. The results from this study present a further understanding of the sophisticated molecular mechanisms of bacterial QS signalling systems.